Circulation of Laguna Negra Virus Associated with Calomys Callosus

HANTAVIRUS PULMONARY SYNDROME IN NORTHWESTERN ARGENTINA: CIRCULATION OF LAGUNA NEGRA VIRUS ASSOCIATED WITH CALOMYS CALLOSUS

SILVANA LEVIS, JORGE GARCIA, NOEMI´ PINI, GLADYS CALDERO´ N, JOSEFINA RAMI´REZ, DANIEL BRAVO, STEPHEN ST. JEOR, CARLOS RIPOLL, MARIANA BEGO, ELENA LOZANO, RUBE´ N BARQUEZ, THOMAS G. KSIAZEK, AND DELIA ENRIA Instituto Nacional de Enfermedades Virales Humanas Dr. Julio I. Maiztegui, Pergamino, Argentina; Hospital San Miguel, Yuto, Jujuy, Argentina; Hospital Oscar Orías, Libertador Gral. San Martín, Jujuy, Argentina; Department of Microbiology, University of Nevada, Reno, Nevada; Dirección de Epidemiología, Jujuy, Argentina; Fundación Miguel Lillo, Tucuman, Argentina; Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, Georgia

Abstract. The purpose of this study was to characterize the hantaviruses circulating in northwestern Argentina. Human and rodent studies were conducted in Yuto, where most cases of hantavirus pulmonary syndrome (HPS) occur. Partial virus genome sequences were obtained from the blood of 12 cases of HPS, and from the lungs of 4 Calomys callosus and 1 Akodon simulator. Phylogenetic analysis showed that three genotypes associated with HPS circulate in Yuto. Laguna Negra (LN) virus, associated with C. laucha in Paraguay, was identified for the first time in Argentina; it was recovered from human cases and from C. callosus samples. The high sequence identity between human and rodent samples implicated C. callosus as the primary rodent reservoir for LN virus in Yuto. The genetic analysis showed that the Argentinian LN virus variant differed 16.8% at the nucleotide level and 2.9% at the protein level relative to the Paraguayan LN virus. The other two hantavirus lineages identified were the previously known Bermejo and Ora´n viruses.

INTRODUCTION In the hantavirus-endemic area of northwestern Argentina, Hantaviruses are rodent-borne viruses that belong to the hantavirus disease was first diagnosed in 1994 in the subtropi- genus Hantavirus of the family Bunyaviridae.1 The hantavi- cal region of Orán, in which HPS case-patients were associ- ruses have tripartite, single-stranded RNA genomes. The ated with viruses of the ORN genotype. After the recognition small (S) genomic segment encodes the nucleocapsid (N) pro- of the disease in the neighboring province of Jujuy in 1997, an tein; the medium (M) genomic segment encodes the glyco- increasing number of HPS cases were prospectively diag- proteins G1 and G2, and the large (L) genomic segment en- nosed by enzyme-linked immunosorbent assay (ELISA). The codes the viral RNA-dependent RNA polymerase.2 After the largest number of HPS cases diagnosed in Jujuy Province identification of Sin Nombre (SN) virus in 1993, many other originated in Ledesma Department (74.6%, 53 of 71); the hantaviruses have been identified in the Americas. Hantavi- main focus was located in the rural town of Yuto, with 62.3% rus pulmonary syndrome (HPS) is a public health problem of (33 of 53) of the HPS cases recorded in this department (Ri- increasing significance, especially in South America, where an poll C, unpublished data). Interestingly, a higher incidence increasing number of HPS cases are reported each year.3,4 of human hantavirus infection and a generally less severe Among the hantaviruses known to cause HPS in South clinical form of HPS has been observed in this location (Pini America are Juquitiba (JUQ),5 Araraquara (ARA), and N and others, unpublished data and Ripoll C, unpublished Castelo dos Sonhos (CAS) viruses from Brazil,6 and Laguna data). This finding is different from the epidemiology of HPS Negra (LN) virus from Paraguay and Bolivia.7 Rio Mamore in the rest of the hantavirus-endemic areas of Argentina. To (RM) virus from Bolivia and Caño Delgadito virus from Ven- determine the genetic diversity of hantaviruses causing HPS ezuela have been detected in the rodent species Oryzomys in northwestern Argentina, we studied the genetic types of microtis and Sigmodon alstoni, respectively, but have not yet hantaviruses found in human and rodent samples from this been linked to HPS.8–10 In Argentina, seven different geno- region. types of hantaviruses have been identified. Four of them have been associated with human disease and include Andes MATERIALS AND METHODS (AND) virus in the southwestern provinces of Rio Negro, Chubut and Neuquén,11 Lechiguanas (LEC) and Hu39694 Rodent samples. Rodent trapping was conducted at nine viruses in the central Buenos Aires and Santa Fe Provinces, sites in Yuto and the surrounding area, both at the presumed and Orán (ORN) virus in northwestern Salta Province.12,13 sites of infection for known HPS case-patients and in nearby The primary rodent hosts for AND and ORN virus genotypes natural habitats, in May 2000 (Pini N and others, unpublished is Oligoryzomys longicaudatus; while LEC and Hu39694 virus data). Lung tissues from hantavirus antibody–positive rodents genotypes have been found in O. flavescens. Three other virus (seven Calomys callosus and four Akodon simulator) were genotypes have also been recognized: Maciel (MAC), Per- examined following reverse transcription (RT) and amplifi- gamino (PGM), and Bermejo (BMJ) viruses;12–14 MAC and cation by polymerase chain reaction (PCR) in a nested as- PGM viruses have been identified from Necromys benefactus say.15 The PCR products were sequenced. and Akodon azarae from central Argentina, respectively; Patient samples. Human blood clots from serologically while BMJ virus genotype was originally recovered only from confirmed cases of acute HPS from Yuto and other locations a single O. chacoensis captured near Orán. All of these han- of Jujuy Province were used as source for virus RNA extrac- taviruses are hosted by rodents of the family Murinae, sub- tion. Samples sent for clinical diagnosis followed the ethical family Sigmodontinae. procedures of the local hospital where patients were assisted. 658 CIRCULATION OF LAGUNA NEGRA VIRUS IN ARGENTINA 659

The protocol studies conducted in Yuto and surrounding ar- Phylogenetic analysis. Phylogenetic analysis was carried eas were reviewed and approved by the institutional ethical out on the multiple nucleotide (partial S and M genome seg- review committee (Comité de Evaluación Ética de Investiga- ments) and amino acid (partial M-G2 genome segment) se- ciones Biomédicas del Instituto Nacional de Enfermedades quence alignments by using maximum parsimony (Phylo- Virales Humanas). genetic Analysis Using Parsimony [PAUP] version 4.0b4a Isolation of total RNA, RT-PCR, and sequence analy- Macintosh computer software programs).17 Phylogenetic sis. Total RNA was extracted from the lungs of hantavirus analysis by maximum parsimony was obtained by the heuris- antibody–positive rodents and blood clots of hantavirus anti- tic search method. The bootstrap support for the results of the body–positive human by using the RNaid (PLUS) kit (Bio phylogenetic analysis was based on 500 replicates. 101 Inc., La Jolla, CA). Amplification of virus RNA was done by a RT-PCR, followed by a nested PCR amplification as RESULTS described previously.15 The DNA product of the nested PCR was excised after electrophoresis from an agarose gel, and Genetic characterization of virus strains. RNA was ex- bands of the predicted size were purified from gels slices using tracted from available lung tissues of 10 hantavirus antibody– a GeneClean kit (Bio 101 Inc.). The nucleotide sequences of positive rodents (six C. callosus and four A. simulator) and these products were determined by the automatic dydeoxy blood clots from 14 HPS case-patients from Yuto and other cycle sequencing technique (Applied Biosystems, Foster City, locations in the Ledesma Department of Jujuy Province (Fig- CA).3 Oligonucleotides primers were designed based on pre- ure 1). Primers used were designed to amplify partial frag- dicted virus-conserved sequence regions to amplify fragments ments of the N protein gene of the S genome segment, and the of M genome segment glycoprotein-encoding region of Ar- G1 and G2 glycoprotein-encoding regions of the M genome gentinian hantavirus genotypes and LN virus from Paraguay. segment. Nested RT-PCR products of the expected size (273 Primers were designed to amplify fragments of the N protein nucleotides) for the S segment were obtained from five han- of the S segment on the basis of conserved hantavirus se- tavirus antibody–positive rodents, and from 12 HPS cases. quences obtained from the GenBank database including LN Hantavirus M-G1 and M-G2 segment cDNAs fragments (328 virus, AF005727; AND virus strain AH-1, AF324901; ARA virus, and 256 nucleotides, respectively) were amplified from seven AF AF307325; BMJ virus, AF482713; LEC virus, AF482714; ORN patient clots, using a set of primers designed to detect Argen- virus, AF482715; and PGM virus, AF482717.7,12 Extended tinian hantavirus M segment sequences. All attempts to am- sequence analysis was done with primers designed based on plify M segment pieces from A. simulator and C. callosus sequence data obtained in this study. Sequences were aligned hantavirus antibody–positive samples, as well as from samples with those of previously described hantaviruses using BioEdit from the remaining seven HPS case-patients using the same version 5.0.6 (North Carolina State University, Raleigh, NC) sets of primers, failed. and the computer software package Clustal W 1.4,16 followed Because of the close geographic relationship between by manual adjustment based on deduced amino acid se- Yuto, Bolivia, and Paraguay, an M-specific set of primers quences. based on sequences of LN virus was designed for the PCR

FIGURE 1. Jujuy Province in Argentina and localities of the origin of cases of hantavirus pulmonary syndrome and rodents positive for antibodies to hantavirus antibody selected for polymerase chain reaction analysis. 660 LEVIS AND OTHERS analysis of these rodent and human samples. Products of the expected size were obtained from one A. simulator, four C. callosus, and five HPS case-patients. Further amplification using newly designed specific primers based on rodent and HPS case sequences was obtained. The phylogenetic analysis of nucleotide sequence differences in these three regions showed the circulation of three hantavirus lineages causing HPS in the limited geographic area studied. The ORN genotype, previously described from human HPS cases and from O. longicaudatus from the nearby location of Orán in Salta province,12 was recovered from six HPS cases from Yuto. In pairwise comparisons against the nucleotide sequence of the homologous region of previously characterized hantaviruses, the 273-nucleotide sequence of S genome segment exhibited 95.6–98.5% nucleotide identity with the ORN genotype from Salta. A similar pattern was seen with the G2 and G1 fragments of the M segment, showing an identity of 93.7–99.6% nucleotides and 92.3–94.2% nucleotides, respectively, when compared with ORN virus. The second genotype identified was BMJ, previously described from a single O. chacoensis from Orán in Salta Prov- ince12; the virus sequence showed a 82.8%, 95.7%, and 94.2% nucleotide sequence identity in 273-, 328-, and 256-nucleotide fragments from the S, M-G1, and M-G2 genome segments, respectively, when compared with the virus sequence from one O. chacoensis from Salta province. This represents the first demonstration that BMJ virus is associated with HPS in Argentina. Finally, LN virus was recovered for the first time in Argentina from HPS cases and rodent samples. Compari- son of the nucleotide sequences detected in five HPS cases, four C. callosus, and one A. simulator showed 83.6–84.2%, 80.2–85.1%, and 81.2–82.0% nucleotide sequence identity for the S, and M-G1, and M-G2 genome fragments, respectively, when compared with the LN virus from Paraguay. Mean sequence variation among the LN virus detected in the human and rodent samples was 0.6%, 2.7%, and 0.9% for the S, M-G1, and M-G2 genome segment fragments, respectively. Phylogenetic analysis. The phylogenetic analysis based on the M-G1 genome segment-encoding region using maximum parsimony (Figure 2) showed that all samples from C. callosus, one A. simulator, and 5 HPS case samples fell into the LN lineage group; 6 HPS case samples fell into the ORN genetic group, and only one sample from an HPS patient grouped FIGURE 2. Phylogenetic tree inferred by maximun parsimony with BMJ virus. The nodes separating LN, ORN, and BMJ analysis of a 258-nucleotide fragment of the medium–glycoprotein 1 (M-G1) genome segment of Laguna Negra (LN), Orán (ORN), and lineages had bootstraps values of 99%, 95%, and 99%, re- Bermejo (BMJ) viruses derived from rodents and hantavirus pulmo- spectively; however, the exact branching order of the Argen- nary syndrome case samples from Yuto, Argentina and other previ- tinian hantaviruses relative to other South American lineages ously characterized hantaviruses. The third base position was re- can not be resolved by the present phylogenetic analysis. moved from the analysis. Bootstrap confidence limits were calculated ARA virus (host unknown) from Brazil is placed together by using 500 repetitions of analysis; values > 50% are indicated at branch points. Vertical branches are for visual purposes only. M seg- with MAC virus (Akondontini-borne virus) from central Ar- ment sequences with the following GenBank accession numbers were gentina, although with a low (52%) bootstrap support. A used in this study: Andes (AND) AH-1, AF324901; Araraquara 1,371-nucleotide sequence of the M-G2 genome segment was (ARA) AF307327; Bayou (BAY) L36930; BMJ strain Oc2253, generated from one representative strain of each of the three AF028025; Castelo dos Sonhos (CAS) AF307326; Hantaan (HTN) 76-118, Y00386; Hu39694, AF028023; Lechiguanas (LEC) strain hantavirus lineages detected in Yuto. The phylogenetic tree Of22819, AF028022; LN AF005728; Maciel (MAC) strain Bo13796, showed a similar topology to the one obtained on the M-G1 AF028027; Oran strain Ol22996, AF028024; Pergamino (PGM) strain genome segment fragment. The three hantavirus lineages Aa14403, AF028028; Prospect Hill (PH) X55129; Puumala (PUU) (LN, ORN, and BMJ) are positioned together with the other Sotkamo strain, X61034; Seoul (SEO) strain SR-11, M34881; Sin ס ס South American hantaviruses, forming a well supported clade Nombre (SNV) CC74, L33684. Cc Calomys callosus;As Ako- -El Moro Can ס Black Creek Canal; ELMC ס don simulator; BCC ס New York hantavirus, strain Rhode Island 1; DOV ס Figure 3); the nodes separating these lineages had yon; NYR) (90%) bootstrap values of 100%, 60%, and 97%, respectively. The Dobrava virus. genetic analysis showed that the LN virus variant recovered CIRCULATION OF LAGUNA NEGRA VIRUS IN ARGENTINA 661

10,000 inhabitants), and a low case-fatality rate (12.76%) associated with a higher percentage of milder illness (Ripoll C, unpublished data). In our serologic studies of rodents captured in Yuto (Pini N and others, unpublished data), we found that the two most frequently trapped sigmodontine rodent species, C. callosus and A. simulator, were positive for antibodies to hantavirus; neither of these species had previously been associated with human hantavirus infections. To identify the hantavirus(es) causing HPS in Jujuy Prov- ince, PCR amplification and sequencing of the appropriate PCR product was conducted in blood samples from seropos- itive HPS cases and rodent samples. Phylogenetic analysis of M and S genome fragments indicated a high genetic diversity of hantaviruses associated with HPS in the limited geographic area of Yuto: the circulation of three previously known hantaviruses (the Paraguayan LN virus, ORN virus, and BMJ virus) was documented. Phylogenetic analysis based on the sequence data of virus genome fragments of the S, M-G1, and M-G2 segments showed that the LN virus lineage, associated with C. laucha (vesper mouse) in Paraguay,7 was associated with C. callosus rodents and HPS cases from the same area of Yuto (more than 600 km from the area in Paraguay where the virus was described). The high sequence identity between human and rodent samples (> 97.3% nucleotide and 100.0% amino acid) implicated C. callosus as the primary rodent reservoir for LN virus circulating in Yuto. Our rodent trapping data indicated that C. callosus, as well as A. simulator, were the most abundant rodent species captured in the area (137 of 327, 38.0% and 140 of 327, 39%); however, C. callosus exhibited the highest prevalence of antibody to hantavirus (7 of 137, 5.1%). These observations correlate with one of the cri- FIGURE 3. Phylogenetic tree inferred by maximun parsimony teria currently accepted in identifying a primary rodent spe- analysis of a 1,371-nucleotide fragment of M-G2 sequences of a rep- cies as a primary rodent host for hantaviruses.18 The phylo- resentative strain of Orán (ORN), and Bermejo (BMJ) viruses de- genetic analysis places LN virus from Paraguay and Yuto rived from hantavirus pulmonary syndrome case samples, and La- guna Negra (LN) virus derived from Calomys callosus samples from together, with a 100% bootstrap support. Yuto, Argentian and other previously characterized hantaviruses. The fact that LN virus strains in Paraguay and Argentina The third base position was removed from the analysis. Bootstrap (difference between them of 16.8% nucleotides and 2.9% confidence limits were calculated by using 500 repetitions of analysis; amino acids) are associated with different species of the same values > 50% are indicated at branch points. Vertical branches are for rodent genus (C. laucha and C. callosus, respectively), sug- visual purposes only. For M segment sequence GenBank accession numbers used in this study and definitions of abbreviations, see Fig- gests the coevolutionary relationship between the viruses ure 2. and that of the corresponding primarily associated rodent reservoirs. Similarly, examples of a pattern of co-speciation was observed in North American rodent genus Peromyscus: SN-like viruses are associated to genetically distinct P. ma- from C. callosus (sample Cc29582) differed 16.8% at the niculatus and P. leucopus.18–20 Our sequence data also indi- nucleotide level and 2.9% at the deduced amino acid level cated that a LN virus strain was derived from one A. simula- relative to the Paraguayan LN virus; while representative vi- tor. Although the serologic data indicated that there were rus strains of ORN (sample Hu50786) and BMJ (sample four positive A. simulator by ELISA, we detected virus se- Hu52635) lineages, showed 7.6%, 4.3% nucleotide differ- quences using the RT-PCR only in one of them. This could ences and 1.1%, −0.5% amino acid differences relative to the represent a LN virus spillover infection from the primary ro- prototype ORN and BMJ viruses, respectively. dent host C. callosus into A. simulator. The pattern of unique hantavirus infection of a primary rodent host includes the DISCUSSION chronic infection of the primary rodent reservoir species, as shown by the ability to consistently amplify hantavirus se- After the confirmation in 1994 of HPS in northwestern quences from serologically positive rodents. Conversely, spill- Argentina (Orán in Salta Province), intensive surveillance ac- over infections are usually charaterized by acute infection tivities were conducted in the region. In 1997, the neighboring with subsequent clearance of virus.18 Thus, the possibility of province of Jujuy had its first indigenous HPS cases. Most of an spillover from C. callosus into A. simulator cannot be cases in this province have originated in or around the town of excluded. Yuto. A particular epidemiologic pattern characterized the The other two genotypes recovered from HPS cases were HPS cases in this area, with a high incidence of illness (1.46/ the previously characterized Oligoryzomys-borne BMJ and 662 LEVIS AND OTHERS

ORN viruses.12 The recovery of a BMJ viral genotype from Received August 5, 2003. Accepted for publication July 15, 2004. one fatal HPS case from Yuto constitutes the first evidence of Acknowledgments: We thank Dr. S. Morzunov and Dr. John Boone its pathogenicity in Argentina; it was previously known only for critical review of the manuscript; Verónica Fasciani for technical from one O. chacoensis in Orán. However, its association with assistance; and Horacio López, GermánO’Duyer, Cesar Polidoro, human disease was predictable because the phylogenetic Enrique Serrrano, Miguel Canchi, Alberto Segobia, Julio Gil, Ber- analysis had grouped the BMJ viral genotype into the sub- nardino Perez, Monica Diaz, and David Flores for their technical field work. clade of the Oligoryzomys-borne viruses, most of them associated with HPS in Argentina. A recent report described the Financial support: This research was supported by the Adminis- association of the BMJ viral genotype to HPS cases in Bo- tración Nacional de Laboratorios e Institutos de Salud (ANLIS), livia.14 The Oran virus genotype remains the most frequent Ministerio de Salud Pública de la Nación, Argentina; Ministerio de Salud Pública de la Provincia de Buenos Aires, Argentina; and Na- cause of HPS, while previous studies showed that it was the tional Institutes of Health grant 1R01 AI-45059. main cause of HPS near Orán.12 Our sequence data identified ORN virus genotype in 50% (6 of 12) of the human samples Authors’ addresses: Silvana Levis, Jorge Garcia, Noemí Pini, Gladys Calderón, and Delia Enria, Instituto Nacional de Enfermedades that were positive by RT-PCR. Curiously, the high frequency Virales Humanas Dr. Julio I. Maiztegui, Monteagudo 2510, 2700 of ORN virus genotype sequences in human case-patient Pergamino, Buenos Aires, Argentina, Telephone: 54-2477-433044, samples does not correlate with low abundance of the rodent Fax: 54-2477-433045, E-mail: [email protected]. Josefina Ramírez species that serves as its putative reservoir (3.3% of the total and Elena Lozano, Hospital San Miguel, 4518 Yuto, Jujuy, Argen- captures) or the lack of detectable hantavirus antibody preva- tina, Telephone 54-3886-498015. Daniel Bravo, Hospital Oscar Orías, San Martin 66, 4512 Libertador Gral. San Martín, Jujuy, Argentina, lence among rodents from the genus Oligoryzomys. It must Telephone 54-3886-42728. Stephen St. Jeor and Mariana Bego, De- be pointed out that our rodent data correspond to a unique partment of Microbiology, Howard Building 320, Room 146, Univer- rodent capture study conducted in Yuto in 2000, while HPS sity of Nevada, Reno, NV 89557, Telephone: 702-784-6161, Fax: 702- case-patient samples included in the present study comprised 784-1620, E-mail: [email protected]. Carlos Ripoll, Direcciónde Epidemiología, San Martin 41, 4600 San Salvador de Jujuy, Jujuy, a four-year period (1998–2001). Moreover, only two of six Argentina, Telephone 54-388-4221300. Rubén Barquez, Fundación ORN viral genotype sequences were identified in human Miguel Lillo, M. Lillo 205, 4000 Tucumán, Argentina, Telephone case-patient samples from Yuto; the remaining four ORN 54-381-4330888. Thomas G. Ksiazek, Special Pathogens Branch, Cen- viral sequences came from HPS cases from Caimancito, ters for Disease Control and Prevention, 1600 Clifton Road, Atlanta, Perico and Fraile Pintado, locations where no rodent trapping GA 30333, Telephone: 404-371-5329, Fax: 404-371-5449. was conducted. As rodent community diversity is influenced Reprint requests: Silvana Levis, Instituto Nacional de Enfermedades by temporal, ecologic, and environmental factors, more in- Virales Humanas Dr. Julio I. Maiztegui, Monteagudo 2510, 2700 Per- tensive rodent serologic surveys in the region would serve to gamino, Buenos Aires, Argentina. clarify this point. Interestingly, one of the major differences in rodent behav- ior observed between Oligoryzomys and Calomys species is REFERENCES the relative capacity of C. callosus to invade human dwellings. 1. 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